Epothilones C, D, E and F, preparation and compositions

ABSTRACT

The present invention relates to epothilones C, D, E and F, their preparation and their use for the production of therapeutic compositions and compositions for plant production.

The present application is a divisional application, claiming priorityto application Ser. No. 10/988,328, filed on Nov. 12, 2004, which is adivision of application Ser. No. 09/313,524, filed on May 17, 1999,which is a continuation of application No. PCT/EP97/06442, filed on Nov.18, 1997, which claims priority to DE 197 07 506.1, filed Feb. 25, 1997and DE 196 47 580.5, filed Nov. 18, 1996.

The present invention relates to epothilones C, D, E and F, theirpreparation and their use for the production of therapeutic compositionsand compositions for plant protection.

Epothilones C and D

According to one embodiment, the invention relates to epothilones [C andD] which are obtainable in that

(a) Sorangium cellulosum DSM 6773 is cultured in a manner known per sein the presence of an adsorber resin,

(b) the adsorber resin is removed from the culture and washed with awater/methanol mixture,

(c) the washed adsorber resin is eluted with methanol and the eluate isconcentrated to give a crude extract,

(d) the concentrate obtained is extracted with ethyl acetate, theextract is concentrated and partitioned between methanol and hexane,

(e) the methanolic phase is concentrated to give a raffinate and theconcentrate is fractionated on a Sephadex column,

(f) a fraction containing metabolic products of the microorganismemployed is obtained,

(g) the fraction obtained is chromatographed on a C18 reverse phase to amethanol/water mixture and, sequentially

after a first fraction containing epothilone A and

a second fraction containing epothilone B

a third fraction containing a first further epothilone and

a fourth fraction containing a second further epothilone are obtainedand

(h1) and the epothilone of the first further fraction and/or

(h2) the epothilone of the second further fraction are isolated.

The invention further relates to an epothilone [C] of the empiricalformula C₂₆H₃₉NO₅S, characterized by the ¹H- and ¹³C-NMR spectrum as inTable 1.

The invention furthermore relates to epothilone C of the formula:

-   Epothilone C R═H

The invention furthermore relates to epothilone [D] of the empiricalformula C₂₇H₄₁NO₅S, characterized by the ¹H- and ¹³C-NMR spectrum as inTable 1.

The invention furthermore relates to epothilone D of the formula:

-   Epothilone D R═CH₃

Epothilones C and D can be used for the preparation of the compounds ofthe following formula 1, where for their derivatization reference can bemade to the derivatization methods described in WO-A-97/19 086.

In the above formula 1:

-   R═H, C₁₋₄-alkyl;-   R¹, R², R³, R⁴, R⁵═H, C₁₋₆-alkyl,    -   C₁₋₆-acylbenzoyl,    -   C₁₋₄-trialkylsilyl,    -   benzyl,    -   phenyl,    -   C₁₋₆-alkoxy-,    -   C₆-alkyl-, hydroxy- and halogen-substituted benzyl or phenyl;        where two of the radicals R¹ to R⁵ can also combine to form the        group —(CH₂)_(n)— with n=1 to 6 and the alkyl or acyl groups        contained in the radicals are straight-chain or branched        radicals;        Y and Z are either identical or different and are each hydrogen,        halogen, such as F, Cl, Br or I, pseudohalogen, such as —NCO,        —NCS or —N₃, OH, O—(C₁₋₆)-acyl, O—(C₁₋₆)-alkyl, O-benzoyl. Y and        Z can also be the O atom of an epoxide, epothilone A and B not        being claimed, or form one of the C—C bonds of a C═C double        bond.

Thus the 12,13-double bond can be selectively

hydrogenated, for example catalytically or with di-imine, a compound ofthe formula 1 being obtained with Y═Z═H; or

epoxidized, for example with dimethyldioxirane or a peracid, a compoundof the formula 1 being obtained with Y and Z═—O—; or

converted into the dihalides, dipseudohalides or diazides, a compound ofthe formula 1 being obtained with Y and Z═Hal, pseudo-hal or N₃.

Epothilones E and F

According to a further embodiment the invention relates to abiotransformant of epothilone A, which is obtainable in that

-   (a) Sorangium cellulosum DSM 6773 is cultured in a manner known per    se in the presence of an adsorber resin, removed from the adsorber    resin and, if appropriate, the total amount or a part of the    separated culture is treated with a methanolic solution of    epothilone A,-   (b) the culture treated with epothilone A is incubated and then    treated with adsorber resin,-   (c) the adsorber resin is separated from the culture, eluted with    methanol and the eluate is concentrated to give a crude extract,-   (d) the crude extract is partitioned between ethyl acetate and    water, the ethyl acetate phase is separated off and concentrated to    give an oil,-   (e) the oil is chromatographed on a reverse phase under the    following conditions:-    column material: Nucleosil 100 C-18 7 μm-    column dimensions: 250×16 mm-    eluent: methanol/water=60:40-    flow rate: 10 ml/min    and fractions having a content of biotransformant and which can be    detected by UV extinction at 254 nm and have an R_(t) value of 20    min are separated off and the biotransformants are isolated.

The invention furthermore relates to a biotransformant of epothilone Aof this type, which is obtainable in that in stage (a) a culture isseparated off which is three or four or more days old.

The invention furthermore relates to a biotransformant of epothilone Aof this type, which is obtainable in that in stage (b) incubation iscarried out for one or two or more days.

The invention furthermore relates to a compound of the empirical formulaC₂₆H₃₉NO₇S, characterized by the following ¹H-NMR spectrum (300 MHz,CDCl₃): delta=2.38 (2-H_(a)), 2.51 (2-H_(b)), 4.17 (3-H), 3.19 (6-H),3.74 (7-H), 1.30-1.70 (8-H, 9-H₂, 10-H₂, 11-H₂), 2.89 (12-H), 3.00(13-H), 1.88 (14-H_(a)), 2.07 (14-H_(b)), 5.40 (15-H), 6.57 (17-H), 7.08(19-H), 4.85 (21-H₂), 1.05 (22-H₃), 1.32 (23-H₃), 1.17 (24-H₃), 0.97(25-H₃), 2.04 (27-H₃)

The invention furthermore relates to a compound (epothilone E) of theformula:

Epothilone E R═H

According to a further embodiment, the invention relates to abiotransformant of epothilone B, which is obtainable in that

-   (a) Sorangium cellulosum DSM 6773 is cultured in a manner known per    se in the presence of an adsorber resin, separated from the adsorber    resin and, if appropriate, the total amount or a part of the    separated culture is treated with a methanolic solution of    epothilone B,-   (b) the culture treated with epothilone B is incubated and then    treated with adsorber resin,-   (c) the adsorber resin is separated from the culture, eluted with    methanol and the eluate is concentrated to give a crude extract,-   (d) the crude extract is partitioned between ethyl acetate and    water, the ethyl acetate phase is separated off and concentrated to    give an oil,-   (e) the oil is chromatographed on a reverse phase under the    following conditions:-    column material: Nucleosil 100 C-18 7 μm-    column dimensions: 250×16 mm-    eluent: methanol/water=60:40-    flow rate: 10 ml/min    and fractions having a content of biotransformant and which can be    detected by UV extinction at 254 nm and have an R_(t) value of 24.5    min are separated off and the biotransformants are isolated.

The invention furthermore relates to a biotransformant of epothilone Bof this type, which is obtainable in that in stage (a) a culture isseparated off which is three or four or more days old.

The invention furthermore relates to a biotransformant of epothilone Bof this type, which is obtainable in that in stage (b) incubation iscarried out for one or two or more days.

The invention furthermore relates to a compound of the empirical formulaC₂₇H₄₁NO₇S, characterized by the following ¹H-NMR spectrum (300 MHz,CDCl₃): delta=2.37 (2-H_(a)), 2.52 (2-H_(b)), 4.20 (3-H), 3.27 (6-H),3.74 (7-H), 1.30-1.70 (8-H, 9-H₂, 10-H₂, 11-H₂), 2.78 (13-H), 1.91(14-H), 2.06 (14-H_(b)), 5.42 (15-H), 6.58 (17-H), 7.10 (19-H), 4.89(21-H₂), 1.05 (22-H₃), 1.26 (23-H₃), 1.14 (24-H₃), 0.98 (25-H₃), 1.35(26-H₃), 2.06 (27-H₃).

The invention furthermore relates to a compound (epothilone F) of theformula:

-   Epothilone F R═CH₃

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a HPLC analysis of an XAD eluate at the end of afermentation.

FIG. 2 shows an enrichment of epothilone E and F in a fermentation brothafter feeding of a mixture of epothilone A and B, analysed afterincubation for 48 hours.

FIG. 3 shows kinetics of the biotransformation of epothilone A toepothilone E by Sorangium cellulosum So ce90.

FIG. 4 shows biotransformation of epothilone A to epothilone E.

FIG. 5 shows biotransformation of epothilone B to epothilone F.

Preparation and Compositions

The compounds or epothilones according to the invention are obtainableby the abovementioned measures.

The invention furthermore relates to compositions for plant protectionin agriculture, forestry and/or horticulture, consisting of one or moreof the abovementioned epothilones C, D, E and F or consisting of one ormore of the abovementioned epothilones in addition to one or morecustomary carrier(s) and/or diluent(s).

The invention finally relates to therapeutic compositions, consisting ofone or more of the abovementioned compounds or one or more of theabovementioned compounds in addition to one or more customary carrier(s)and/or diluent(s). In particular, these compositions can show cytotoxicactivities and/or bring about immunosuppression and/or be employed forthe control of malignant tumours, it being particularly preferablypossible for them to be used as cytostatics.

In the following, the invention is illustrated and described in greaterdetail by the description of some selected working examples.

EXAMPLES Example 1 Epothilones C and D

A. Production Strain and Culture Conditions According to the EpothiloneBasic Patent DE-B-41 38 042.

B. Production with DSM 6773

75 1 of culture are grown as described in the basic patent and used forthe inoculation of a production fermenter with 700 1 of productionmedium consisting of 0.8% starch, 0.2% glucose, 0.2% soya flour, 0.2%yeast extract, 0.1% CaCl₂×2H₂O, 0.1% MgSO₄×7H₂O, 8 mg/l of Fe-EDTA,pH=7.4 and optionally 15 l of Amberlite XAD-16 adsorber resin. Thefermentation lasts 7-10 days at 30 C, aeration with 2 m³/hr. Bycontrolling the speed of rotation, the pO₂ is kept at 30%.

C. Isolation

The adsorber resin is separated from the culture using a 0.7 m², 100mesh process filter and freed from polar concomitants by washing with 3bed volumes of water/methanol 2:1. By elution with 4 bed volumes ofmethanol, a crude extract is obtained which is evaporated in vacuo untilthe water phase appears. This is extracted three times with the samevolume of ethyl acetate. Evaporation of the organic phase affords 240 gof crude extract, which is partitioned between methanol and heptane inorder to separate off lipophilic concomitants. By evaporation in vacuo,180 g of raffinate are obtained from the methanol phase and arefractionated into three portions on Sephadex LH-20 (column 20×100 cm, 20ml/min of methanol). The epothilones are contained in the fraction of atotal of 72 g eluted with a 240-300 min retention time. To separate theepothilones, the fraction is chromatographed in three portions onLichrosorb RP-18 (15 μm, column 10×40 cm, eluent 180 ml/minmethanol/water 65:35). After epothilone A and B, epothilone C, withR_(t)=90-95 min, and epothilone D, 100-110 min, are eluted and afterevaporation in vacuo obtained in a yield of 0.3 g each as colourlessoils.D. Physical Properties

-   Epothilone C R═H-   Epothilone D R═CH₃    Epothilone C-   C₂₆H₃₉NO₅S [477]-   ESI-MS: (positive ions): 478.5 for [M+H]⁺-   1H and 13C see NMR table-   TLC: R_(f)=0.82-   TLC aluminum foil 60 F 254 Merck, eluent:    dichloromethane/methanol=9:1-   Detection: UV extinction at 254 nm. Spraying with vanillin-sulphuric    acid reagent, blue-grey coloration on heating to 120° C.-   HPLC: R_(t)=11.5 min-   Column: Nucleosil 100 C-18 7 μm, 125×4 mm-   Eluent: methanol/water=65:35-   Flow rate: 1 ml/min-   Detection: diode array    Epothilone D-   C₂₇H₄₁NO₅S [491]-   ESI-MS: (positive ions): 492.5 for [M+H]⁺-   1H and 13C see NMR table-   TLC: R_(f)=0.82-   TLC aluminum foil 60 F 254 Merck, eluent:    dichloromethane/methanol=9:1-   Detection: UV extinction at 254 nm. Spraying with vanillin-sulphuric    acid reagent, blue-grey coloration on heating to 120° C.-   HPLC: R_(t)=15.3 min-   Column: Nucleosil 100 C-18 7 μm, 125×4 mm-   Eluent: methanol/water=65:35-   Flow rate: 1 ml/min

Detection: diode array TABLE 1 ¹H- and ¹³C-NMR data of epothilone C andepothilone D in [D₆] DMSO at 300 MHz Epothilone C Epothilone D H atom δ(ppm) C atom δ (ppm) δ (ppm) C atom δ (ppm) 1 170.3 1 170.1 2-Ha 2.38 238.4 2.35 2 39.0 2-Hb 2.50 3 71.2 2.38 3 70.8 3-H 3.97 4 53.1 4.10 453.2 3-OH 5.12 5 217.1 5.08 5 217.4 6-H 3.07 6 45.4 3.11 6 44.4 7-H 3.497 75.9 3.48 7 75.5 7-OH 4.46 8 35.4 4.46 8 36.3 8-H 1.34 9 27.6 1.29 929.9 9-Ha 1.15 10 30.0 1.14 10 25.9 9-Hb 1.40 11 27.6 1.38 11 31.8*10-Ha 1.15* 12 124.6 1.14* 12 138.3 10-Hb 1.35* 13 133.1 1.35* 13 120.311-Ha 1.90 14 31.1 1.75 14 31.6* 11-Hb 2.18 15 76.3 2.10 15 76.6 12-H5.38** 16 137.3 16 137.2 13-H 5.44** 17 119.1 5.08 17 119.2 14-Ha 2.3518 152.1 2.30 18 152.1 14-Hb 2.70 19 117.7 2.65 19 117.7 15-H 5.27 20164.2 5.29 20 164.3 17-H 6.50 21 18.8 6.51 21 18.9 19-H 7.35 22 20.87.35 22 19.7 21-H₃ 2.65 23 22.6 2.65 23 22.5 22-H₃ 0.94 24 16.7 0.90 2416.4 23-H₃ 1.21 25 18.4 1.19 25 18.4 24-H₃ 1.06 27 14.2 1.07 26 22.925-H₃ 0.90 0.91 27 14.1 26-H₃ 1.63 27-H₃ 2.10 2.11*, **assignment interchangeable

Example 2 Epothilone A and 12,13-bisepi-epothilone A from Epothilone C

50 mg of epothilone A are dissolved in 1.5 ml of acetone and treatedwith 1.5 ml of a 0.07 molar solution of dimethyldioxirane in acetone.After standing at room temperature for 6 hours, the mixture isevaporated in vacuo and the residue is separated by preparative HPLC onsilica gel (eluent: methyl tert-butyl ether/petroleum ether/methanol33:66:1).

Yield:

-   25 mg of epothilone A, R_(t)=3.5 min (analyt. HPLC, 7 μm, column    4×250 mm, eluent see above, flow rate 1.5 ml/min)    and-   20 mg of 12,13-bisepi-epothilone A, R_(t)=3.7 min, ESI-MS (pos.    ions) m/e=494 [M+H]⁺

¹H-NMR in [D₄] methanol, selected signals: delta=4.32 (3-H), 3.79 (7-H),3.06 (12-H), 3.16 (13-H), 5.54 (15-H), 6.69 (17-H), 1.20 (22-H), 1.45(23-H).

-   12,13-bisepi-epothilone A R═H

Example 3 Epothilone E and F, Novel Biotransformation Products ofEpothilones A and B

Production Strain:

The production strain Sorangium cellulosum So ce90 was isolated in July1985 in GBF from a soil sample from the banks of the Zambesi anddeposited on 28.10.91 in the German Collection for Microorganisms underNo. DSM 6773.

The characterization of the producer and the culture conditions aredescribed in:

Höfle, G.; N. Bedorf, K. Gerth & H. Reichenbach: Epothilones, processesfor their preparation and compositions containing them. DE 41 38 042 A1,laid open on 27 May 1993.

Formation of Epothilones E and F During Fermentation:

A typical fermentation proceeds in the following manner: A 100 lbioreactor is filled with 60 l of medium (0.8% starch; 0.2% glucose;0.2% soya flour; 0.2% yeast extract; 0.1% CaCl₂×2H₂O; 0.1% MgSO₄×7H₂O; 8mg/l of Fe-EDTA; pH 7.4). 2% of adsorber resin (XAD-16, Rohm & Haas) areadditionally added. The medium is sterilized by autoclaving (2 hours,120° C.). Inoculation is carried out with 10 l of a preculture grown inthe same medium (additionally 50 mM HEPES buffer pH 7.4) in a shakerflask (160 rpm, 30° C.). Fermentation is carried out at 32° C. with astirrer speed of 500 rpm and an introduction of 0.2 N1 per m³ per hourof air, the pH is kept at 7.4 by addition of KOH. The fermentation lasts7 to 10 days. The epothilones formed are continuously bound to theadsorber resin during the fermentation. After separating off the culturebroth (e.g. by screening in a process filter), the resin is washed with3 bed volumes of water and eluted with 4 bed volumes of methanol. Theeluate is concentrated to dryness and taken up in 700 ml of methanol.

HPLC Analysis of the XAD Eluate:

In relation to the starting volume of the reactor (70 l), the eluate isconcentrated 100:1. The analysis is carried out using a 1090 HPLC unitfrom Hewlett Packard. To separate the constituents, a microbore column(125/2 Nucleosil 120-5 C₁₈) from Machery-Nagel (Düren) is used. Elutionis carried out using a gradient of water/acetonitrile from initially75:25 up to 50:50 after 5.5 minutes. This ratio is maintained up to the7th minute, in order to then increase it up to the 10th minute to 100%acetonitrile.

Measurement is carried out at a wavelength of 250 nm and a bandwidth of4 nm. The diode array spectra are measured in the wavelength range from200 to 400 nm. In the XAD eluate, two novel substances with R_(t) 5.29and R_(t) 5.91 stand out, whose adsorption spectra are identical withthose of epothilones A and B (FIG. 1; E corresponds to A, F correspondsto B). These substances are only formed in traces under the givenfermentation conditions.

Biotransformation of Epothilone A and B to Epothilone E and F:

A 500 ml culture of So ce90, 4 days old and maintained with adsorberresin, is used for the specific biotransformation. 250 ml of this aretransferred to a sterile 1 l Erlenmeyer flask leaving behind the XAD. Amethanolic solution of a mixture of a total of 36 mg of epothilone A+14mg of B is then added and the flask is incubated on a shaking rack fortwo days at 30° C. and 200 rpm. The formation of the epothilones E and Fis analysed directly from 10 μl of the centrifuged culture supernatant(FIG. 2). The conversion takes place only in the presence of the cellsand is dependent on the cell densities employed and the time. Kineticsof the conversion are shown for epothilone A in FIG. 3.

Isolation of Epothilone E and F

To isolate epothilone E and F, three shaker flask batches from thebiotransformation (see above) are combined and shaken with 20 ml ofXAD-16 for 1 h. The XAD is obtained by screening and eluted with 200 mlof methanol. The eluate is evaporated in vacuo to give 1.7 g of crudeextract. This is partitioned between 30 ml of ethyl acetate and 100 mlof water. On evaporation in vacuo, 330 mg of an oily residue areobtained from the ethyl acetate phase, which are chromatographed in fiveruns on a 250×20 mm RP-18 column (eluent: methanol/water 58:42,detection 254 nm).

Yield: epothilone E 50 mg

-   -   F 10 mg        Biological Action of Epothilone E:

In cell cultures, the concentration was determined which reduces thegrowth by 50% (IC₅₀) and compared with the values for epothilone A. IC₅₀(ng/ml) Cell line Epothilone E Epothilone A HeLa.KB-3.1 (human) 5 1Mouse fibroblasts, L929 20 4Epothilone E

-   C₂₆H₃₉HO₇S [509]-   ESI-MS: (positive ions): 510.3 for [M+H]⁺-   TLC: R_(f)=0.58-   TLC aluminum foil 60 F 254 Merck, eluent:    dichloromethane/methanol=9:1-   Detection: UV extinction at 254 nm. Spraying with vanillin-sulphuric    acid reagent, blue-grey colouration on heating to 120° C.-   HPLC: R_(t)=5.0 min-   Column: Nucleosil 100 C-18 7 μm, 250×4 mm-   Eluent: methanol/water=60:40-   Flow rate: 1.2 ml/min-   Detection: diode array    ¹H-NMR (300 MHz, CDCl₃): delta=2.38 (2-H_(a)), 2.51 (2-H_(b)), 4.17    (3-H), 3.19 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H₂, 10-H₂, 11-H₂),    2.89 (12-H), 3.00 (13-H), 1.88 (14-H_(a)), 2.07 (14-H_(b)), 5.40    (15-H), 6.57 (17-H), 7.08 (19-H), 4.85 (21-H₂), 1.05 (22-H₃), 1.32    (23-H₃), 1.17 (24-H₃), 0.97 (25-H₃), 2.04 (27-H₃)    Epothilone F-   C₂₇H₄₁NO₇S [523]-   ESI-MS: (positive ions): 524.5 for [M+H]⁺-   TLC: R_(f)=0.58-   TLC aluminum foil 60 F 254 Merck, eluent:    dichloromethane/methanol=9:1-   Detection: UV extinction at 254 nm. Spraying with vanillin-sulphuric    acid reagent, blue-grey colouration on heating to 120° C.-   HPLC: R_(t)=5.4 min-   Column: Nucleosil 100 C-18 7 μm, 250×4 mm-   Eluent: methanol/water=60:40-   Flow rate: 1.2 ml/min-   Detection: diode array    ¹H-NMR (300 MHz, CDCl₃): delta=2.37 (2-H_(a)), 2.52 (2-H_(b)), 4.20    (3-H), 3.27 (6-H), 3.74 (7-H), 1.30-1.70 (8-H, 9-H₂, 10-H₂, 11-H₂),    2.78 (13-H), 1.91 (14-H), 2.06 (14-H_(b)), 5.42 (15-H), 6.58 (17-H),    7.10 (19-H), 4.89 (21-H₂), 1.05 (22-H₃), 1.26 (23-H₃), 1.14 (24-H₃),    0.98 (25-H₃), 1.35 (26-H₃), 2.06 (27-H₃).

Example 4 Preparation of Epothilone E and F by Biotransformation withSorangium cellulosum So ce90

1) Carrying out the Biotransformation:

For the biotransformation, a culture of Sorangium cellulosum So ce90 isused which has been shaken for four days in the presence of 2% XAD 16adsorber resin (Rohm and Haas, Frankfurt/M.) at 30° C. and 160 rpm. Theculture medium has the following composition in g/litre of distilledwater: potato starch (Maizena), 8; glucose (Maizena), 8; defatted soyaflour, 2; yeast extract (Marcor), 2; ethylenediaminetetraacetic acid,iron(III) sodium salt, 0.008; MgSO₄×7H₂O, 1; CaCl₂×2 H₂O, 1; HEPES 11.5.The pH is adjusted to 7.4 before autoclaving with KOH. The XAD isseparated from the culture by sieving through a stainless steel sieve(200 μm mesh width). The bacteria are sedimented by centrifugation for10 min at 10,000 rpm and the pellet is resuspended in ⅕ of the culturesupernatant. Epothilone A or epothilone B in methanolic solution is thenadded to the concentrated bacterial suspension in a concentration of 0.5g/ litre. The culture is cultured further as described above. To analysethe biotransformation, a 1 ml sample is taken at the desired times, 0.1ml of XAD is added and the sample is shaken at 30° C. for 30 min. TheXAD is eluted with methanol. The eluate is concentrated to dryness andtaken up again in 0.2 ml of methanol. This sample is analysed by meansof HPLC. FIG. 4) Kinetics of the biotransformation of epothilone A toepothilone E FIG. 5) Kinetics of the biotransformation of epothilone Bto epothilone F.

2) Preparation of Epothilone E by Biotransformation of 1 g of EpothiloneA.

The strain Sorangium cellulosum So ce90 is grown for four days in 8.5 lof the above medium (but without XAD addition) in a 10 litre bioreactorat 30° C., a speed of rotation of 150 rpm and an introduction of 1 l/minof air.

The culture is then concentrated to 3 l by crossflow filtration. Forthis purpose, 0.6 m² of a membrane having a pore size of 0.3 μm areused.

The concentrated culture is transferred to a 4 litre bioreactor and amethanolic solution of 1 g of epothilone A in 10 ml of methanol isadded. The culture is then cultured further for a period of time of 21.5h. The temperature is 32° C., the stirrer speed is 455 rpm and theintroduction of air takes place at 6 l/min. At the time of harvesting,100 ml of XAD is added and the mixture is incubated further for 1 h. TheXAD is separated from the cells by screening and exhaustively elutedwith methanol. The concentrated eluate is analysed by means of HPLC.

Balancing of the Biotransformation:

-   Epothilone A employed: 1000 mg=100%-   Epothilone A recovered after 21.5 h: 53.7 mg=5.4%-   Epothilone E formed after 21.5 h: 661.4 mg=66.1%-   Epothilone A completely decomposed:=28.5%

Experiment 5

The epothilones according to the invention were tested with cellcultures (Table 2) and for promotion of polymerization (Table 3). TABLE2 Epothilone tests with cell cultures Epothilone B C A 507 477 D E F 493IC-50 [ng/ml] 491 509 523 Mouse fibroblasts L 4 1 100 20 20 1.5 929human tumor cell lines: HL-60 (leukaemia) 0.2 0.2 10 3 1 0.3 K-562(leukaemia) 0.3 0.3 20 10 2 0.5 U-937 (lymphoma) 0.2 0.2 10 3 1 0.2KB-3.1 (carcinoma 1 0.6 20 12 5 0.5 of the cervix) KB-V1 (carcinoma 0.30.3 15 3 5 0.6 of the cervix multires) A-498 (carcinoma — 1.5 150 20 203 of the kidney) A-549 (carcinoma 0.7 0.1 30 10 3 0.1 of the lung)

TABLE 3 Polymerization test with epothilones Parameter: Time up to thehalf-maximal polymerization of the control Agent Agent Measurement: w xy z [s] [%] Control 200 170 180 210 190 100 Epothilone A 95 60 70 70 7439 Epothilone B 23 25 30 26 14 Epothilone C 125 76 95 80 94 49Epothilone D 125 73 120 106 56 Epothilone E 80 60 50 45 59 31 EpothiloneF 80 40 30 50 50 26Standard Test with 0.9 mg of Tubulin/ml and 1 μM Sample Concentration

The polymerization test is an in vitro test using purified tubulin frompigs' brain. Evaluation is carried out photometrically.Polymerization-promoting substances such as the epothilones reduce thetime up to which half-maximal polymerization has taken place, i.e. theshorter the time, the more active the compound. w, x, y and z are fourindependent experiments, the relative activity is expressed in the lastcolumn in % of the control; again the lowest values indicate the bestactivity. The ranking list corresponds reasonably accurately to thatfound in cell cultures.

1-16. (canceled)
 17. A compound of the formula:

said compound having a state of purity such as to be substantially freeof other major metabolic products produced by Sorangium cellulosum. 18.A composition comprising at least one compound according to claim 1 andat least one carrier or diluent.
 19. A method of treating malignanttumors comprising administering to a recipient in need thereof atherapeutic amount of a compound of the formula